In the third year of this project, we have continued with the analysis of viral genetic determinants involved in the pathogenesis of the acutely lethal SIVsmmPBj isolate. Completion of our studies with the nef gene have confirmed that the tyrosine-17 residue of Nef is required for acute pathogenesis. Additionally, mutation of this residue affected the ability of virus to replicate in macrophages--a key pathogenic determinant in PBj-induced disease. However, the mutation did not affect the ability of the virus to grow in unstimulated peripheral blood mononuclear cells (PBMC) even when depleted of macrophages. In vivo the virus did not induce acute disease, but still appeared to induce lymphocyte homing and lymphoid hyperplasia in the gut area. We have generated several new chimeric and deletion mutants of our PBj6.6 clone. A vif/vpx chimera, prepared by substituting the SIVsmm9 vif/vpx area for that of PBj6.6 did not change the in vitro phenotype of the virus, suggesting that these determinants are not important in the overall pathogenic effects of PBj. A deletion mutant of the vif gene in our PBj6.6 clone was generated, but turned out to be inactive, confirming that vif is required for viral infectivity. We have constructed a chimeric clone containing the region coding for the matrix protein of SIVsmH4 to investigate the possible role of this protein in PBj pathogenesis. Finally, we have generated a chimeric clone which contains the tat gene of SIVsmH4. In vitro analysis of both of these clones is about to commence. To investigate the early events in the growth of this virus in the macaque, we have initiated a serial time-course study. Pig-tailed macaques were inoculated with SIVsmmPBj and sacrificed on days 1-6 post infection. Analysis of plasma samples from these animals showed that infectious virus could not be recovered until 2 days post infection. Titers increased as time progressed. Infectious virus could be recovered from PBMC as early as day 1 post infection. Similar to results with plasma, titers increased with time after infection. We are currently focusing on cells obtained from tissue samples for virus titration, but this may not give a good indication of viral load and localization because of possible blood contamination. Analysis of cytokines in plasma is ongoing.